High-dimensional mass cytometry analysis of NK cell ......2020/10/01 · 3 INTRODUCTION Natural...
Transcript of High-dimensional mass cytometry analysis of NK cell ......2020/10/01 · 3 INTRODUCTION Natural...
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Key points:
• A disruption in the maturation process of NK cells leads to accumulation of unconventional CD56-
CD16+ NK cells in patients with AML
• High frequency of CD56-CD16+ NK cells is associated with adverse clinical outcome
AML group 1
AML group 2
Healthy subjects
AML group 1
AML group 2
Healthy subjects
AML group 1
CD
56
CD16
AML group 2
NK cells
% O
S
Time from diagnosis (months)
Early stages Late stages
NK cell maturation
0 20 40 60 80 1000
20
40
60
80
100
CD56
NKG2A
CD158a,h
CD16 CD57
CD158b1,b2,j Bifurcation
point
High-dimensional mass cytometry analysis of NK cell alterations in Acute Myeloid
Leukemia identifies a subgroup with adverse clinical outcome
Anne-Sophie Chretien, Raynier Devillier, Samuel Granjeaud, Charlotte Cordier, Clemence Demerle, Nassim
Salem, Julia Wlosik, Florence Orlanducci, Emilie Gregori, Magali Paul, Philippe Rochigneux, Thomas
Pagliardini, Mathieu Morey, Cyril Fauriat, Nicolas Dulphy, Antoine Toubert, Herve Luche, Marie Malissen,
Didier Blaise, Jacques A. Nunès, Norbert Vey, and Daniel Olive.
Correspondence
Graphical abstract
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NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
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High-dimensional mass cytometry analysis of NK cell alterations in Acute
Myeloid Leukemia identifies a subgroup with adverse clinical outcome
Anne-Sophie Chretien1,2
, Raynier Devillier1-3
, Samuel Granjeaud4, Charlotte Cordier
1,2,5, Clemence Demerle
1,2,
Nassim Salem1,2
, Julia Wlosik1,2
, Florence Orlanducci1,2
, Emilie Gregori6, Magali Paul
7, Philippe Rochigneux
1,2,8,
Thomas Pagliardini1-3
, Mathieu Morey9, Cyril Fauriat
1,2, Nicolas Dulphy
10, Antoine Toubert
10, Herve Luche
6,
Marie Malissen6,11
, Didier Blaise1,3
, Jacques A. Nunès1, Norbert Vey
3, and Daniel Olive
1,2.
Author affiliations
1Team Immunity and Cancer, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068, CNRS, UMR7258, Institut Paoli-
Calmettes, Aix-Marseille University, UM105, Marseille, France; 2Immunomonitoring department, Institut Paoli-Calmettes, Marseille,
France; 3Hematology Department, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm, U1068, CNRS, UMR7258, Institut
Paoli-Calmettes, Aix-Marseille University, UM 105, Marseille, France; 4Systems Biology Platform, Centre de Recherche en Cancérologie de
Marseille (CRCM), Inserm, U1068, CNRS, UMR7258, Institut Paoli-Calmettes, Aix-Marseille University, UM 105, Marseille, France; 5Biopathology Department, Institut Paoli-Calmettes, Marseille, France;
6Centre d'Immunophénomique–Luminy (Ciphe), Inserm US012,
CNRS UMS3367, Aix-Marseille University, Marseille, France; 7ImCheck therapeutics, Marseille, France;
8Medical Oncology Department,
Institut Paoli-Calmettes, Marseille, France; 9Datactivist, Aix-en-Provence, France;
10Université Paris Diderot, Sorbonne Paris Cité, Institut
Universitaire d'Hématologie, Immunology and Histocompatibility department, Hôpital Saint-Louis, APHP, Inserm UMRS-1160, Paris, France;
11Centre d'Immunologie de Marseille-Luminy (CIML), Inserm CNRS, Aix-Marseille University, Marseille, France.
Corresponding author:
Pr. Daniel Olive
Immunomonitoring Platform
Paoli-Calmettes Institute
Team Immunity and Cancer
Centre de Recherche en Cancérologie de Marseille (CRCM)
Inserm U1068
27 Boulevard Lei Roure CS 30059
13273 Marseille Cedex 09 - FRANCE
Phone: +33 486 977 271
Running title: CD56-CD16
+ NK cells in AML
Key words: AML, natural killer cells, CD56-CD16
+ NK cells, prognostic biomarkers, mass cytometry
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ABSTRACT
Natural killer (NK) cells are major anti-leukemic immune effectors. Leukemic blasts have a negative
impact on NK cell function and promote the emergence of phenotypically and functionally impaired
NK cells. In the present work, we highlight an accumulation of CD56-CD16
+ unconventional NK cells in
acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients
chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood
from patients with newly-diagnosed AML (N=48, HEMATOBIO cohort, NCT02320656) and healthy
subjects (N=18) by mass cytometry. We evidenced a moderate to drastic accumulation of CD56-
CD16+ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of
NKG2A as well as the triggering receptors NKp30, and NKp46, in line with previous observations in
HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased
expression of three additional major triggering receptors required for NK cell activation, NKG2D,
DNAM-1, and CD96. A high proportion of CD56-CD16
+ NK cells at diagnosis was associated with an
adverse clinical outcome, with decreased overall survival (HR=0.13; P=.0002) and event-free survival
(HR=0.33; P=.018), and retained statistical significance in multivariate analysis. Pseudo-time analysis
of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation
from conventional NK cells toward CD56-CD16
+ NK cells. Overall, our data suggest that the
accumulation of CD56-CD16
+ NK cells may be the consequence of immune escape from innate
immunity during AML progression.
Significance
This work provides the first report of accumulation of unconventional CD56-CD16+ NK cells in non-
virally induced malignancies. Pseudotime analysis highlights a bifurcation point occurring during the
course of NK cell maturation, providing elements regarding the possible origin of CD56-CD16+ NK
cells. Increased frequency of CD56-CD16+ NK cells is associated with adverse clinical outcome in AML
and might contribute, as well as other maturation defects, to a defective control of AML progression.
Overall, accumulation of CD56-CD16+ NK cells could be an important feature of immune escape from
innate immunity.
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INTRODUCTION
Natural killer (NK) cells are critical cytotoxic effectors involved in leukemic blast recognition, tumor
cell clearance, and maintenance of long-term remission.1 NK cells directly kill target cells without
prior sensitization, enabling lysis of cells stressed by viral infections or tumor transformation. NK cells
are divided into different functional subsets according to CD56 and CD16 expression.2–4
CD56bright
NK
cells are the most immature NK cells found in peripheral blood. This subset is less cytotoxic than
mature NK cells and secretes high amounts of chemokines and cytokines such as IFNγ and TNFα.
These cytokines have a major effect on the infected or tumor target cells, and play a critical role in
orchestration of the adaptive immune response through dendritic cell activation. CD56dim
CD16+ NK
cells, which account for the majority of circulating human NK cells, are the most cytotoxic NK cells.
NK cell activation is finely tuned by integration of signals from inhibitory and triggering receptors, in
particular NKp30, NKp46, and NKp44, DNAM-1, and NKG2D.5 Upon target recognition, CD56
dimCD16
+
NK cells release perforin and granzyme granules and mediate antibody-dependent cellular
cytotoxicity through CD16 (FcɣIIIR) to clear transformed cells.
NK cells are a major component of the anti-leukemic immune response, and NK cell alterations have
been associated with adverse clinical outcomes in acute myeloid leukemia (AML).6–9
Therefore, it is
crucial to better characterize AML-induced NK cell alterations in order to optimize NK cell-targeted
therapies. During AML progression, NK cell functions are deeply altered, with decreased expression
of NK cell-triggering receptors and reduced cytotoxic functions, as well as impaired NK cell
maturation.6,9–13
Cancer-induced NK cell impairment occurs through various mechanisms of immune
escape, including shedding and release of ligands for NK cell-triggering receptors, release of
immunosuppressive soluble factors such as TGFβ, adenosine, PGE2, or L-kynurenine, and
interference with NK cell development, among others.14
Interestingly, these mechanisms of immune evasion are also seen to some extent in chronic viral
infections, notably HIV.2 In patients with HIV, NK cell functional anergy is mediated by the release of
inflammatory cytokines and TGFβ, the presence of MHClow
target cells, and the shedding of ligands
for NK cell-triggering receptors.2 As a consequence, some phenotypical alterations described in
cancer patients are also induced by chronic HIV infections, with decreased expression of major
triggering receptors such as NKp30, NKp46, and NKp44,15,16
decreased expression of CD16,17
and
increased expression of inhibitory receptors such as TIGIT18
all observed. In addition, patients with
HIV display an accumulation of CD56-CD16
+ unconventional NK cells, a highly dysfunctional NK cell
subset.19,20
Mechanisms leading to the loss of CD56 are still poorly described, and the origin of this
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subset of CD56- NK cells is still unknown. To date, two hypotheses have been considered: CD56
- NK
cells could be terminally differentiated cells arising from a mixed population of mature NK cells with
altered characteristics, or could expand from a pool of immature precursor NK cells.21
Expansion of
CD56-CD16
+ NK cells is mainly observed in viral non-controllers.
19,20 Indeed, CD56 is an important
adhesion molecule involved in NK cell development, motility and pathogen recognition.22–27
CD56 is
also required for the formation of the immunological synapse between NK cells and target cells, lytic
functions, and cytokine production.26,28
As a consequence, CD56-CD16
+ NK cells display lower
degranulation capacities, decreased expression of triggering receptors, perforin, and granzyme B,
dramatically reducing their cytotoxic potential, notably against tumor target cells.2,19,20,29,30
In line
with this loss of the cytotoxic functions against tumor cells, patients with concomitant Burkitt
lymphoma and Epstein Barr virus infection display a dramatic increase of CD56-CD16
+ NK cells,
30
which could represent an important hallmark of escape to NK cell immunosurveillance in virus-driven
hemopathies.
To our knowledge, this population has not been characterized in the context of non-virally induced
hematological malignancies. In the present work, we investigated the presence of this population of
unconventional NK cells in patients with AML, its phenotypical characteristics, and the consequences
of its accumulation on disease control. Finally, we explored NK cell developmental trajectories
leading to the emergence of this phenotype.
PATIENTS AND METHODS
Patients and study design
This monocentric study (Paoli-Calmettes Institute, Marseille, France) included forty-eight patients
with newly diagnosed non-acute promyelocytic leukemia AML from the HEMATOBIO cohort
(NCT02320656). Patients were aged 19 to 80 years (mean ±SD = 54.0±14.0). All patients received
cytarabine and anthracycline–based induction chemotherapy as previously described.43
Eight
patients (16.7%) were treated with allogeneic stem cell transplantation as a consolidation therapy.
Patients’ characteristics are summarized in Table 1.
Ethics statement
All participants gave written informed consent in accordance with the Declaration of Helsinki. The
entire research procedure was approved by the institutional review board of the Paoli-Calmettes
Institute.
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Clinical samples
Peripheral blood mononuclear cells (PBMCs) cryopreserved in 90% albumin/10% DMSO were
obtained before (N=48) and after induction chemotherapy (N=16, paired samples), and from age-
matched healthy volunteers (N=18). Handling, conditioning, and storage of samples were performed
by the Paoli-Calmettes Tumor bank, which operates under authorization # AC-2007-33 granted by
the French Ministry of Research.
Mass cytometry analysis
PBMCs were thawed and processed as previously described.6 PBMCs were washed with RPMI 1640
medium with 10% fetal calf serum and incubated in RPMI 1640 with 2% fetal calf serum and 1/10000
Pierce® Universal Nuclease 5kU (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2
for 30 min. Cells were centrifuged and incubated with cisplatin 0.1 M to stain dead cells. Aspecific
epitopes were blocked with 0.5 mg/mL Human Fc Block (BD Biosciences, San Jose, CA, USA). Two
million PBMCs were stained for 45 min at 4°C with the extracellular antibodies (Supplemental Table
1). Cells were centrifuged and barcoded with the Cell-ID™ 20-Plex Pd Barcoding Kit (Fluidigm, San
Francisco, CA, USA) according to the manufacturer's recommendations. Cells were washed and
samples were combined and stained with metal-labeled anti-phycoerythrin secondary antibodies for
30 min at 4°C. After centrifugation, cells were washed and permeabilized with the Foxp3 Staining
Buffer Set (eBioscience, San Diego, CA, USA) for 40 min at 4°C. Intracellular aspecific epitopes were
blocked with 0.5 mg/mL Human Fc Block for 40 min at 4°C before incubation with the mix of
intracellular antibodies for 40 min at 4°C in Foxp3 Staining Buffer (Supplemental Table 1). Cells were
then washed and labeled overnight with 125 nM iridium intercalator (Fluidigm) in Cytofix (BD
Biosciences). Finally, cells were diluted in EQTM Four Element Calibration Beads (Fluidigm) before
acquisition on a Helios® instrument (Fluidigm).
Statistical analysis
Statistical analyses were carried out using Graph Pad Prism V5.01 (San Diego, CA, USA) and SPSS V9.0
(Chicago, IL, USA). The χ2 or Fisher’s exact test was used to assess association between variables. For
multiple comparisons of paired values, a Friedman test was performed followed by a Dunn’s post-
test. Groups of patients with a high or low frequency of CD56-CD16
+ NK cells were defined based on
optimized cut-points using maximally selected log-rank statistics (maxstat package, R software V
3.6.2)44
and on imposing that no group could represent fewer than 25% of patients. For survival
analyses, overall survival (OS) was defined as the time from diagnosis until death from any cause, and
event-free survival (EFS) as the time between induction and relapse, death from any cause, or
induction failure, whatever occurred first. Patients without an event were censored at the time of
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their last follow-up. Survival times were estimated by the Kaplan–Meier method and compared using
the log-rank test. A multivariate Cox regression model was used to assess the prognostic value of
CD56-CD16
+ NK cells while adjusting for other prognostic factors. Candidate variables for the Cox
regression were European Leukemia Net genetic classification, age at diagnosis, leukocytosis, and
percentage of CD56-CD16
+ NK cells. Continuous variables were discretized as follows: age < or ≥50
years old; leukocytosis: < or ≥50 G/L; CD56-CD16
+ NK cells: < or ≥10%. All factors with a P value
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The frequency of CD56-CD16
+ NK cells was significantly higher in group 2 than in group 1 or in healthy
volunteers, with mean frequencies of 24.9% vs 3.5% and 7.7%, P
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cells were also present in healthy volunteers (Figure 2C and 3). Furthermore, CD56-CD16
+ NK cells
displayed reduced expression of the anti-apoptotic proteins BCL-2 and BCL-XL compared with
CD56dim
CD16+ NK cells in both healthy volunteers and patients with AML (Figure 4).
Evolution at complete remission
When material was available, we then assessed variations of the frequency of CD56-CD16
+ NK cells at
complete remission compared with baseline frequencies (Supplemental Figure 4). A normal
frequency of CD56-CD16
+ NK cells was not systematically restored after induction chemotherapy.
After induction chemotherapy, 7 out of 16 patients (43.8%) displayed less than 10% of CD56-CD16
+
NK cells. Among them, 5 patients maintained long-term complete remission and 2 patients relapsed.
Nine out of 16 patients (56.2%) displayed more than 10% of CD56-CD16
+ NK cells after induction
chemotherapy. Among them, 3 patients maintained long-term complete remission and 6 patients
relapsed.
High frequency of CD56-CD16
+ NK cells at diagnosis is associated with adverse clinical outcome
Patients were classified into two groups according to the frequency of CD56-CD16
+ NK cells, using the
thresholds of 10% defined above. The frequency of CD56-CD16
+ NK cells at diagnosis in patients with
AML was analyzed according to clinical outcome after 24 months of follow-up. After induction
therapy, complete remission rates were 82.9% and 69.2% in groups 1 and 2, respectively. Among
patients without accumulation of CD56-CD16
+ NK cells (group 1), 18 out of 35 (54.3%) patients were
in continuous CR after 24 months of follow-up, whereas among patients with accumulation of CD56-
CD16+ NK cells (group 2), 2 out of 13 patients were in continuous CR (15.4%) (Figure 5A). Overall
survival (OS) (hazard ratio (HR)=0.13, P
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maturation using the Wishbone algorithm. Wishbone is a recent algorithm used for analysis of
developmental pathways in high-dimensional single-cell datasets. This algorithm positions single cells
along bifurcating developmental trajectories on a k-nearest neighbor graph and pinpoints bifurcation
points.32
The rise and fall of markers acquired or lost during the course of NK cell development (y
axis) is represented as a function of pseudo-time (x axis) (Figure 6A and B). Each branch after the
bifurcation point represents a distinct differentiation trajectory. Wishbone recovers hallmarks of NK
cell maturation in healthy volunteers. NK cells initially highly express CD56 and NKG2A. CD56bright
NK cells expressing low levels of CD16 correspond to a transition between early immature
CD56bright
CD16− NK cells and CD56
dimCD16
+ NK cells. Subsequently, NK cells lose expression of NKG2A
and sequentially acquire KIRs (CD158a,h and CD158b1,b2,j) and finally CD57, which marks the
acquisition of high cytotoxic potential4 (Figure 6A). In AML, the maturation process is disrupted and
leads to a bifurcation point: the first branch displays a normal-like maturation profile, whereas the
second branch displays altered maturation features, with decreased CD57 and CD158b1,b2,j
expression, as well as loss and re-acquisition of CD16 (Figure 6B). These observations further support
the hypothesis of a bifurcation from conventional NK cells toward CD56-CD16
+ NK cells during the
maturation process.
DISCUSSION
Recent clinical trials highlight the central role of NK cells for the control of AML.33
Since NK cell
alterations are expected to impact response to NK cell-based immunotherapies, a better
characterization of evasion from NK cell surveillance is therefore an absolute prerequisite to design
the next-generation of therapeutic strategies based on NK cell manipulation. In the present article,
we highlight the presence of unconventional CD56- NK cells in 10% of patients with AML, which is
associated with adverse clinical outcomes. This variable appears to be an independent prognostic
factor since it retains statistical significance in multivariate analysis. However, allogeneic stem cell
transplantation was not equally distributed within groups 1 and 2, with 7 out of 35 patients (20%) vs
1 out of 13 (7.7%) receiving allo-SCT, respectively. This difference was not significant and could be
partially explained by the lower complete remission rates in group 2.
CD56- NK cells represent a minor subset of NK cells under physiological conditions. Their relative
proportion increases in chronically infected HIV viremic patients, in which the CD56-CD16
+ subset can
represent up to 50% of total NK cells.19,20,34,35
More recently, this population has been described in
subjects co-infected with cytomegalovirus (CMV) and Epstein-Barr virus (EBV), with a moderate
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increase in frequency.36
Elevated frequencies of CD56- NK cells have also been described to a lesser
extent in other viral chronic infections, such as hepatitis C or hantavirus infections, 37,38
and auto-
immune ocular myasthenia.39
In these pathological situations, functional characterization of these
CD56− NK cells revealed profound dysfunctions. In HIV-infected subjects, CD56
-CD16
+ NK cells are
reported to be highly dysfunctional, with reduced natural cytotoxicity, defective antibody-dependent
cellular cytotoxicity, as well as low IFNγ production capacity, although they retain chemokine
secretion capacities.20,21
Phenotypical characterization of CD56-CD16
+ NK cells highlighted a
decreased expression of the NK cell-triggering receptors NKp30 and NKp46, as well as a decreased
expression of NKG2A.20
Whether this population of NK cells is induced by chronic exposure to
antigens is unclear, and mechanisms involved in CD56-CD16
+ NK cell accumulation remain to be
elucidated. Overall, previous reports suggest that these NK cells represent a subset of exhausted NK
cells.20,21,35
Interestingly, an important downregulation of CD56 could be obtained in vitro using sera
from patients with chronic lymphoid leukemia added to NK cells from healthy subjects, suggesting
that this phenotype emerges in the presence of an immunosuppressive milieu.40
Beside the question of the mechanisms involved in the emergence of CD56-CD16
+ NK cells, the
important question of the origin of this subset remained to be explored. We used a pseudo-time
algorithm to explore differentiation trajectories that give rise to CD56-CD16
+ NK cells. These
algorithms were developed to investigate the fundamental questions of development processes from
individual cells into different cell types. Interestingly, we could reconstitute the theoretical scheme of
NK cell maturation in healthy subjects. Identification of a bifurcation occurring during the course of
NK cell maturation in patients with AML provides answers regarding the possible origin of CD56-
CD16+ NK cells, suggesting that accumulation of CD56
- NK cells would be the consequence of
disruption of the maturation process. This observation provides bases for further exploration of the
mechanisms involved in the emergence of this population, which will enable to better define
therapeutic strategies likely to restore physiological frequencies of CD56- NK cells. These elements
must be considered with regards to other maturation anomalies our group described in AML, with a
maturation blockade that affects approximately 10% of patients, for whom the clinical outcome is
dramatic.6 As with maturation blockade, bifurcation toward CD56
- NK cells has major consequences
on clinical outcome. Hence, a normal NK cell maturation appears to be critical to control the disease
and these anomalies urgently need to be further explored in AML. Interestingly, it has been reported
that this phenotype is reversed when the viral load of patients with HIV was lowered below
detectable levels after 12 months of antiretroviral therapy, a situation associated with considerable
improvement in NK cell function, increased NK cell receptor expression, and restoration of normal
CD56 expression.21
In the context of AML, the normalization of the frequency of CD56- NK cells was
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observed only in some patients at the time of complete remission. This persistence of CD56- NK cells
30 days after induction chemotherapy should be explored at later time points to confirm these
results.
To our knowledge, accumulation of CD56- NK cells has not been reported in hematological or solid
malignancies, except in chronic natural killer cell large granular lymphocytosis (LGL), where there is a
massive clonal expansion of CD56dim/-
NK cells in a subset of patients.41
According to the authors,
CD56- NK cell expansion in LGL leukemia might be the consequence of an activating stimulus such as
a retroviral infection, notably because sera from patients with LGL leukemia frequently react with
HTLV-I/II p21 envelope proteins. Whether CMV infection could induce this aberrant phenotype has
not been determined in our cohort, as we do not have CMV status data for all our patients. However,
the increase in CD56- NK cells described in CMV and EBV co-infected subjects is moderate
36 and
therefore does not explain the massive accumulation we observe in AML, although a synergistic
mechanism cannot be excluded. Finally, we observed a higher prevalence of CD56- NK cell
accumulation in AML with inversion 3 (inv 3); in 3 out of 4 patients, CD56-CD16
+ NK cells represented
more than 20% of the total NK cell population. This association might represent an interesting
feature of immune alteration in the group of patients with inv 3 for whom clinical outcome is
extremely unfavorable, and requires confirmation in a larger cohort of patients.42
In conclusion, loss of CD56 expression by the NK cells of patients with AML might represent a feature
of immune evasion from NK cell control. If the impact of such a phenotype on clinical outcome is
confirmed, the frequency of CD56- NK cells at diagnosis might be informative in NK cell-based
immune signatures to predict clinical outcome in AML. Lastly, exploration of the mechanisms
involved in downregulation of CD56 will provide new opportunities to restore NK cell functions in this
specific subgroup of patients.
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ACKNOWLEDGMENTS
The authors thank the CRCM cytometry core facility as well as the IPC/CRCM/UMR 1068 Tumor Bank,
which operates under authorization # AC-2007-33 granted by the French Ministry of Research
(Ministère de la Recherche et de l’Enseignement Supérieur). Louise Ball from Angloscribe, an
independent scientific language editing service, provided drafts and editorial assistance to the
authors during preparation of this manuscript. This work has been financially supported by the INCa
(grant 2012-064/2019-038 for D.O., and A.T.), the Fondation de France (grant 00076207 for A.S.C),
the SIRIC Marseille (grant INCa-DGOS-INSERM 6038), the Cancéropôle PACA (grants K_CyTOF 2014
and AML_CyTOF 2016 for J.A.N.), the GS IBiSA and the Agence Nationale de la Recherche
(PHENOMIN project for M.M., H.L., and E.G.). The team “Immunity and Cancer” was labeled “Equipe
FRM DEQ 201 40329534” (for D.O.).
AUTHORSHIP
Contribution:
C.C., C.D., E.G., F.O., M.P., and N.S. performed experiments
A.-S.C., H.L., J.W., M.M., P.R., and S.G., analyzed results and prepared the figures
A.-S.C., A.T., C.F., D.B., D.O., H.L., J.A.N., M.M., N.V., R.D., and T.P. designed the research and wrote
the article
Conflict-of-interest disclosure:
The authors declare no relevant conflicts of interest
Correspondence: Daniel Olive, Immunomonitoring Platform, Paoli-Calmettes Institute, Team
Immunity and Cancer, Centre de Recherche en Cancérologie de Marseille (CRCM), Inserm U1068, 27
Boulevard Lei Roure CS 30059, 13273 Marseille Cedex 09, France; [email protected]
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted October 2, 2020. ; https://doi.org/10.1101/2020.10.01.20204867doi: medRxiv preprint
https://doi.org/10.1101/2020.10.01.20204867http://creativecommons.org/licenses/by-nc-nd/4.0/
-
13
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https://doi.org/10.1101/2020.10.01.20204867http://creativecommons.org/licenses/by-nc-nd/4.0/
-
4.4%
16.3% 76.4%
2.7%
HV#75C
D5
6 1
76
Yb
CD16 209Bi
0.9%
16.8% 79.7%
2.5%
AML#17
0.5%
16.3% 64.0%
18.7%
AML#30
0%
26.9%
33.6%
39.2%
AML#26 AML#13
0.6%
7.4%11.2%
80.8%
AML#36
2.5%
6.3% 89.1%
2.0%
HV
AML group 2
AML group 1
A
CD56bright NK cells
CD56dimCD16− NK cells
CD56dimCD16+ NK cells
CD56−CD16+ NK cells
1.7%
16.0% 78.1%
3.2%
7.7%
14.2% 74.1%
4.5%
HV#71HV#76
AML#12
3.8%
11.8% 84.2%
1.4%
0 20 40 60 80
Ke
rne
ld
en
sity
% CD56−CD16+ NK cells
HV
AML group 1
AML group 2
HV
AML
grou
p1
AML
grou
p2
0
20
40
60
80
100
**
*
***
%C
D5
6-
CD
16
+
B C
%C
D5
6−C
D1
6+
NK
ce
lls
Figure 1. Accumulation of unconventional CD56-CD16+ NK cells in AML. PBMC from 48 newly-diagnosed AML patients and 18
HV were phenotyped by mass cytometry. (A) NK cell phenotype by CD56 and CD16 expression; representative examples of HV
and AML patients without (group 1) or with (group 2) accumulation of CD56-CD16+ NK cells. (B) Frequency of CD56-CD16+ NK
cells in HV and patients with AML. Results are presented as interquatile ranges, median, and whiskers from minimum to
maximum. (C) Threshold visualization and value distribution illustrated by Kernel density estimation. HV, healthy volunteer. *:
P
-
2
4
8
5
117
1
3
10
6
9
AML group 2AML group 1HV
NKG2A NKp30 NKp46
NKG2D DNAM-1 CD96
A
B
Clusters #
CD56 CD16
HighLow
Cellular
density
High
Low
Marker
expression
High
Low
CD4
BcL-XL
Ki-67
CD27
DNAM-1
NKp46
CD96
BCL-2
NKG2C
CD56
NKG2D
NKG2A
CD57
CD158a,h
CD158b1,b2,j
CD45RA
NKp30
CD16
CD8
7 10 3 11 8 5 4 6 9 1 2
Marker expression
Figure 2. Loss of NK cell-triggering
receptors in CD56-CD16+ NK cells. (A)
total NK cells from peripheral blood were
manually pre-gated and exported in
Cytosplore for h-SNE analysis. Consensus
files were generated for each group of
patients with fixed number of NK cells.
Left panel: h-SNE enables identification of
NK cell clusters based on CD56 and CD16
expression; right panel: the heatmap
summarizes phenotypical characteristics
of NK cell populations identified by h-SNE.
(B) Expression of NK cell-triggering
receptors projected on h-SNE maps. (C)
Left panel: NK cell-triggering receptor
expression profiles in clusters of
CD56bright, CD56dimCD16-, CD56dimCD16+,
and CD56-CD16+ NK cells were confirmed
by manual gating to enable quantification
of differences between clusters of NK
cells. Results are presented as interquatile
ranges, median, and whiskers from
minimum to maximum. Data were
analyzed using a Friedman test followed
by a Dunn’s test. Right panel: marker
expression by NK cell subset; blue:
CD56bright NK cells; orange: CD56dimCD16-
NK cells; grey: CD56dimCD16+ NK cells;
green: CD56-CD16+ NK cells. *: P
-
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
*ns
**
*nsns
% N
Kp
30+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
***ns
***
ns*ns
% N
Kp
46+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
***ns
***
**ns
% N
Kp
46+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
***ns
***
***nsns
% N
Kp
30+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
**ns
***
**nsns
% D
NA
M-1
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
**ns
***
***ns
% N
KG
2D+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
***ns
***
***nsns
% N
KG
2D+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
***ns
***
****ns
% D
NA
M-1
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+
0
20
40
60
80
100
**ns
***
*nsns
% C
D96
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+
0
20
40
60
80
100
****
***
*nsns
% C
D96
+ N
K c
ells
NKp30
HV AML
NKp46
NKG2D
DNAM-1
CD96
C
Figure 2 (continuation)
17
6Y
b C
D5
6
169Tm NKp30
17
6Y
b C
D5
6
162Dy NKp46
17
6Y
b C
D5
6
166Er CD96
17
6Y
b C
D5
6
160Gd NKG2D
17
6Y
b C
D5
6
164Dy DNAM-1
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted October 2, 2020. ; https://doi.org/10.1101/2020.10.01.20204867doi: medRxiv preprint
https://doi.org/10.1101/2020.10.01.20204867http://creativecommons.org/licenses/by-nc-nd/4.0/
-
NKG2A
HV AML
CD57
CD158a,h
CD158b1,b2,j
17
6Y
b C
D5
6
154Sm CD158b1,b2,j
17
6Y
b C
D5
6
168Er CD158a,h
17
6Y
b C
D5
6
172Yb CD57
17
6Y
b C
D5
6
165Ho NKG2ACD
56br
ight
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
****
nsns*
% N
KG
2A+
NK
cel
ls
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
***ns
******
ns
% C
D57
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
***ns
****
ns
% C
D15
8a,h
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
**ns
******ns
% C
D15
8b1,
b2,
j+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
**ns
ns***
ns
% C
D15
8b1,
b2,
j+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
***ns
ns*
ns
% C
D15
8a,h
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns
***ns
**nsns
% C
D57
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns**
***
*ns***
% N
KG
2A+
NK
cel
ls
Figure 3. CD56-CD16+ NK cell clusters display intermediate maturation profiles. Left panel: expression of
maturation markers in clusters of CD56bright, CD56dimCD16-, CD56dimCD16+, and CD56-CD16+ NK cells was
analyzed by manual gating. Results are presented as interquatile ranges, median, and whiskers from
minimum to maximum. Differences between clusters were analyzed using a Friedman test followed by a
Dunn’s test. Right panel: marker expression by NK cell subset; blue: CD56bright NK cells; orange: CD56dimCD16-
NK cells; grey: CD56dimCD16+ NK cells; green: CD56-CD16+ NK cells. *: P
-
BCL-2
HV AML
BCL-XL
17
6Y
b C
D5
6
158Gd BCL-XL
17
6Y
b C
D5
6
150Nd BCL-2
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
nsns
*
*ns***
% B
CL-
XL
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
ns*
ns
***ns*
% B
CL-
XL
+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
nsns
***
***nsns
% B
clL
2+ N
K c
ells
CD56
brig
ht
CD56
dim
CD1
6-
CD56
dim
CD1
6+
CD56
- CD1
6+0
20
40
60
80
100
*ns
***
***nsns
% B
clL
2+ N
K c
ells
Figure 4. CD56-CD16+ NK cells display decreased expression of anti-apoptotic proteins. Left panel:
expression of anti-apoptotic proteins in clusters of CD56bright, CD56dimCD16-, CD56dimCD16+, and CD56-CD16+
NK cells was analyzed by manual gating. Differences between clusters were assessed using a Friedman test
followed by a Dunn’s test. Results are presented as interquatile ranges, median, and whiskers from
minimum to maximum. Right panel: marker expression by NK cell subset; blue: CD56bright NK cells; orange:
CD56dimCD16- NK cells; grey: CD56dimCD16+ NK cells; green: CD56-CD16+ NK cells. *: P
-
%C
D5
6−C
D1
6+
NK
ce
lls
A
CR group
Relapse group
Refractory goup
B% CD56 −CD16+ NK cells ≤10
% CD56 −CD16+ NK cells >10
0 20 40 60 80 1000
20
40
60
80
100
Time from induction (months)
% E
FS
0 20 40 60 80 1000
20
40
60
80
100
Time from diagnosis (months)
% O
S
P=.0002
HR (CI95) = 0.13 (0.05-0.38)
P=.018
HR (CI95) = 0.33 (0.13-0.83)
Figure 5. High frequency of CD56-CD16+ NK cells at diagnosis is associated with adverse clinical outcome.
(A) Frequency of CD56-CD16+ NK cells in AML patients at diagnosis according to clinical outcome after 24
months of follow-up. (B) Patients were stratified according to the frequency of CD56-CD16+ NK cells (group
1: CD56-CD16+ NK cells ≤10%; group 2: CD56-CD16+ NK cells >10%). The impact of the accumulation of
CD56-CD16+ NK cells on OS and EFS was assessed using a Log rank test. CI95, 95% confidence interval; CR,
complete remission; HR, hazard ratio; and UPN, unique patient number.
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted October 2, 2020. ; https://doi.org/10.1101/2020.10.01.20204867doi: medRxiv preprint
https://doi.org/10.1101/2020.10.01.20204867http://creativecommons.org/licenses/by-nc-nd/4.0/
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172Yb CD57 154Sm 158b1,b2,j 152Sm NKG2C 209Bi CD16 168Er CD158a,h
165Ho NKG2A 176Yb CD56 Wishbone trajectory Branch association
172Yb CD57 154Sm 158b1,b2,j 152Sm NKG2C 209Bi CD16
165Ho NKG2A 176Yb CD56 Wishbone trajectory Branch association
168Er CD158a,h
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Wishbone trajectory
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Figure 6. Differentiation trajectories are distinct for CD56dimCD16+ NK cells and CD56-CD16+ NK cells. NK cells were manually pre-gated and
exported for differentiation trajectory inference using the Wishbone algorithm in HV (A) and AML patients at diagnosis (B). Wishbone enables
identification of the branch point that gives rise to the population of CD56-CD16+ NK cells. Ordering and branching of cells along bifurcating
developmental trajectories was performed using t-distributed stochastic neighbor embedding (t-SNE) and diffusion maps. The resulting
trajectory and branches are used to visualize the dynamics of maturation markers (CD56, CD57, NKG2A, and KIRs) and of CD16 during NK cell
differentiation.
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted October 2, 2020. ; https://doi.org/10.1101/2020.10.01.20204867doi: medRxiv preprint
https://doi.org/10.1101/2020.10.01.20204867http://creativecommons.org/licenses/by-nc-nd/4.0/